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Detection of Respiratory Viruses with Plastic High Throughput Screening Devices

机译:用塑料高通量筛选装置检测呼吸道病毒

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Microarrays have become one of the most convenient tools for high throughput screening and have catalyzed major advances in genomics and proteomics. Other important applications can be found in medical diagnostics, detection of biothreats, drug discovery, etc. Integration of microarrays with microfluidic devices can be highly advantageous in terms of portability, shorter analysis time and lower consumption of expensive biological analytes. Since fabrication of microfluidic devices using traditional materials such as glass is rather expensive, there is a high interest in employing polymeric materials as a low cost alternative suitable for mass production. We present proof-of-concept DNA arrays on a plastic platform for the detection of four important human respiratory pathogens: influenza A virus, respiratory syncytial virus, enterovirus, and metapneumovirus. This was accomplished by amplifying the genetic material from the viruses and simultaneously labeling the amplicons with a fluorescent dye (Cy3) via a highly sensitive multiplex Reverse Transcription Polymerase Chain Reaction (RT-PCR). The resultant RT-PCR product was hybridized, without further purification, with an array of specific oligonucleotide probes (20 mers) that had been covalently bound to a plastic substrate. Results indicate a high signal to background ratio that is comparable to commercially available microarray glass slides. In addition, 5 minute hybridization on this plastic substrate has been demonstrated using a centrifugal microfluidic platform, paving the way to a rapid medical diagnostic device for point-of-care use that is based on a low-cost portable Micro-Total-Analysis-System (μ-TAS).
机译:微阵列已成为高吞吐量筛选最方便的工具之一,并催化了基因组学和蛋白质组学的主要进步。其他重要应用可以在医学诊断中发现,检测生物素,药物发现等。微阵列与微流体装置的集成在可移植性方面可以是非常有利的,较短的分析时间和昂贵的生物分析物的较低消耗。由于使用玻璃如玻璃等传统材料的微流体装置的制备相当昂贵,因此在采用聚合物材料作为适合于批量生产的低成本替代物的高兴趣。我们在塑料平台上呈现概念验证DNA阵列,用于检测四种重要人类呼吸道原理:流感病毒,呼吸道和血症病毒,肠病病毒和颅叶血病毒。这是通过从病毒中扩增遗传物质并同时通过高度敏感的多重逆转录聚合酶链反应(RT-PCR)与荧光染料(Cy3)同时标记扩增子。将得到的RT-PCR产物杂交,无需进一步纯化,具有与塑料基材共价结合的特定寡核苷酸探针(20mer)。结果表明与市售的微阵列玻璃载玻片相当的背景比的高信号。此外,使用离心微流体平台已经证明了5分钟的杂交,用于铺平到快速医疗诊断装置,以获得基于低成本便携式微量分析的护理点使用系统(μ-tas)。

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